Imidazoline receptor binding compounds

ABSTRACT

Pentamidine and analogs thereof have activity as imidazoline receptor binding compounds. A method of binding the imidazoline receptor, comprises contacting a bis-benzene to said imidazoline receptor in an amount effective to bind to said receptor, wherein said bis-benzene contains at least one amidine group (e.g., one or two). The contacting step may be carried out in vivo or in vitro. Contacting may be carried out with individual active compounds or with libraries of active compounds.

FIELD OF THE INVENTION

[0001] The present invention concerns compounds that have high affinityfor the imidazoline receptor, particularly the imidazoline I2 receptor,methods of use thereof, and combinatorial libraries thereof.

BACKGROUND OF THE INVENTION

[0002] In the early 1980's while studying the different hypotensiveeffects of imidazoline compounds vs. catecholamines in the nucleusreticularis lateralis, Bousquet et al. proposed the existence of a classof imidazoline (1) binding sites distinct from adrenergic receptors(Bousquet, P. et al., The Journal of Pharmacology and ExperimentalTherapeutics 230:232-236 (1984)).

[0003] Many subsequent studies have supported the presence of thesebinding sites in a variety of tissues (Coupry, I. et al., Biochemical &Biophysical Research Communications 147:1055-1060 (1987); Ernsberger, P.R. et al, European Journal of Pharmacology 134:1-13 (1987); Meeley, M.P. et al., Life Sciences 38:1119-1126 (1986); Parini, A. et al. Journalof Biological Chemistry 264(20):11874-8 (1989)), including the existenceof the putative endogenous Iigand (Atlas, D., and Y. Burstein, EuropeanJournal of Biochemistry 144:287-293 (1984); Li, G. et al., Science263:966-969 (1994)) and purification of a mitochondrial imidazolinebinding site (Limon, I. et al., Journal of Biological Chemistry267(30):21645-9 (1992)).

[0004] The binding sites are classified into two groups, I₁ and I₂,according to their affinity for clonidine and idazoxan, respectively,and are located on the plasma and outer mitochondrial membranes. I₂binding sites are associated with the monoamine oxidase protein incertain tissues and are further sub-classified into I_(2A) and I_(2B)depending on their sensitivity to the compound amiloride (Regunathan,S., and D. J. Reis, Annual Review of Pharmacology & Toxicology 36:511-44(1996)). A third subgroup classified as I_(atypical) have beenidentified in the pancreatic i-cell (Morgan, N. G. et al., Annals of theNew York Academy of Sciences 763:361-73 (1995); Brown, C. A. et al.,British Journal of Pharmacology 108:312-317 (1993); Chan, S. L.,Clinical Science 85(6):671-7 (1993)).

[0005] The I₁ binding sites are localized on the plasma membrane andhave been found in the brainstem, kidneys, adrenal chromaffin cells, ratPC12 cells and platelets. Imidazoline I₁ receptor agonists whichstimulate I₁ receptors in the medulla offer a new therapeutic approachto the treatment of hypertension and associated metabolic syndromecomprising of hyperglycemia, hyperinsulinemia, hypertriglyceridemia andlow concentrations of high-density lipoprotein cholesterol (Krentz, A.J., and A. J. Evans, The Lancet 351:152-153 (1998)). Selectivity forthis receptor reduces the adverse effects attributed to stimulation ofα₂-adrenoceptors Such as sedation and dry mouth. Platelet I₁ receptorsare upregulated in patients suffering with depression and can bedownregulated with antidepressant therapy.

[0006] I₂ binding sites are more widely distributed than I₁ sites. Theyhave been found in the nervous system (cerebral cortex, astrocytes,carotid bodies), specific organs (kidneys, liver, pancreas, colon,urethra, placenta), glands (adrenal medulla, prostate) and certain celltypes (platelets, adipocytes, vascular cells). Expression of I₂ bindingsites is upregulated in human brains during aging, and in patients withAlzheimer's disease. Expression is modified in the brains of depressedsuicide victims (Regunathan, S., and D. J. Reis, Annual Review ofPharmacology & Toxicology 36:511-44.(1996)).

[0007] Efforts have been made to identify compounds with imidazolinereceptor binding affinity:

[0008] U.S. Pat. No. 5,726,197 to Clark et al. describe isoindolinylderivatives that have imidazoline receptor binding affinity.

[0009] U.S. Pat. No. 5,686,477 to Jarry et al. describes various5-(aryloxymethyl) oxazolines having imidazoline binding activity.

[0010] U.S. Pat. No. 5,354,769 to Garcia Sevilla et al. describesbenzofuranyl imidazole derivatives having imidazoline receptor bindingactivity, and various uses thereof.

[0011] U.S. Pat. No. 5,732,717 to Watanabe et al. describes the use of4-chloro-5-(imidazoline-2-ylamio)-6-methoxy-2-methylpyrimidine fortreating substance abuse withdrawal.

[0012] U.S. Pat. No. 5,574,059 to Regunathan et al. describes thetreatment of vascular smooth muscle cell proliferation with certainimidazoline receptor agonists.

[0013] Nevertheless, there are relatively few compounds with imidazolinereceptor binding affinity available, and there is continued interest inidentifying additional imidazoline receptor binding compounds.

SUMMARY OF THE INVENTION

[0014] A first aspect of the present invention is a method of bindingthe imidazoline receptor, comprising contacting a bis-benzene to saidimidazoline receptor in an amount effective to bind to said receptor,wherein said bis-benzene contains at least one amidine group. Theamidine group is covalently bound to one of the benzene rings, or anamidine group is covalently bound to both of the benzene rings (suchcompounds and their pharmaceutically acceptable salts are referred to as“active agents” herein). The contacting step may be carried out in vitro(e.g., with cells that express the imidazoline receptor, or with acell-free preparation comprising the imidazoline receptor) or in vivo(e.g., by administering said compound to a subject afflicted with adisease state which is alleviable by treatment with a compound havinghigh selectivity and affinity for the imidazoline receptor site).

[0015] A second aspect of the present invention is a method ofidentifying imidazoline receptor binding agents, comprising the steps ofproviding a library of bis-benzene compounds, said bis-benzene compoundcontaining at least one amidine group as described above; and screeningsaid library for compounds that bind to said imidazoline receptor. Thelibrary may be a combinatorial library, and the bis-benzene compoundsmay be immobilized on a solid support in accordance with knowntechniques (e.g., where only one of the benzene groups has an amidinegroup covalently bound thereto, the other benzene group may be affixed,e.g., by covalent bond, to the solid support).

[0016] The present invention is explained in greater detail in thedrawings and specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1. Inhibition of 5 nM [³H]idazoxan (K_(d) 14±2nM) binding torat liver membranes by α₂-adrenoceptor ligands and pentamidine.Cirazoline, K_(i)=3.0±0.4 nM; Phentolamine, Ki>5000 nM; Epinephrine,K_(i)>25,000 nM; Yohimbine, Ki>25,000 nM; Pentamidine, K_(i)=1.4±0.22Nm.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Definitions.

[0019] As used herein:

[0020] The term “loweralkyl,” as used herein, refers to C₁-C₆ linear orbranched alkyl, such as methyl, ethyl, propyl, , isopropyl, butyl,sec-butyl, iso-butyl, tert-butyl, pentyl, isopentyl, and hexyl. Isoalkylgroups, such as isopropyl, isobutyl, isopentyl, and the like arecurrently preferred. The term “loweralkoxy” or “oxyalkyl” as usedherein, refers to C₁-C₆ linear or branched alkoxy, such as methoxy,ethoxy, propyloxy, butyloxy, isopropyloxy, and t-butyloxy. Methoxy iscurrently preferred.

[0021] “Cycloalkyl” means a saturated monovalent monocyclic hydrocarbonradical containing 3-8 carbon atoms, such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0022] The term “halo” or “halogen” as used herein refers halogens suchas to Cl, Br, I, F, preferably Cl or Br.

[0023] “Imidazoline receptor” as used herein refers to any imidazolinereceptor, including I₁ and I₂ receptors. The I₂ receptors (includingI_(2A) and I_(2B) receptors) are currently preferred. The imidazolinereceptor may be of any species of origin, but is preferably mammalian(e.g., human, mouse, rat, cat, dog, rabbit, hamster, monkey, etc.).

[0024] “Phenyl” as used herein encompasses all possible isomeric phenylradicals optionally monosubstituted or disubstituted with a substituentselected from the group consisting of lower alkyl, lower alkoxy, andhalo.

[0025] “Pharmaceutically acceptable acid addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

[0026] “[³H]-idazoxan” refers to tritiated idazoxan, named as(1,4-[6,7-3H]benzodioxan-2-yl)-2-imidazoline hydrochloride, or [³H]-RX781094. It is available from Amersham plc.

[0027] The term “treatment” as used herein covers any treatment of adisease in a mammal, particularly a human, and includes: (i) preventingthe disease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (ii) inhibiting thedisease, i.e., arresting its development; or (iii) relieving thedisease, i.e., causing regression of the disease.

[0028] The term “disease state which is alleviable by treatment with acompound having high selectivity and/or affinity for the imidazolinereceptor site” as used herein is intended to cover all disease stateswhich are generally acknowledged in the art to be usefully treated withcompounds having high selectivity and affinity for the imidazoline sitein general, as described in U.S. Pat. No. 5,726,197 to Clark (see column4, lines 1-19 therein). Such disease states include, but are not limitedto, cerebral ischemia, hypertension, excessive intraocular pressure,parkinsonian disorders, eating disorders, seasonal affective disorders,panic disorders, urinary incontinence, diuresis, fertility disorders(including the treatment of infertiliby by, for example, in vitrofertilization, and use in antifertility), sexual dysfunction, impotence,postnatal depression, mild stress-induced amenorrhoea, andgalactorrrhoea, by administering to a mammal in need thereof atherapeutically effective amount of a compound as described herein, or apharmaceutically acceptable salt thereof. In addition, such diseasestates include depression, Alzheimer's disease and diabetes.

[0029] Active compounds

[0030] Active compounds of the present invention are, in general,pentamidine and analogs thereof. Numerous such compounds are known. Inthe present invention both benzene rings need not be substituted with anamidine group: only a single ring need be substituted with an amidinegroup (though optionally both rings may be substituted with an amidinegroup). Such compounds are known or can be synthesized in accordancewith known techniques (See, e.g., U.S. Pat. Nos. 5,723,495; 5,723,288;5,686,456; 5,668,167; 5,667,975; 5,643,935; 5,639,755; 5,627,184;5,622,955; 5,606,058; 5,602,172; 5,594,138; 5,578,631; 5,521,189;5,428,051; 5,206,236; 5,202,320; 4,963,589; 4,940,723; 4,933,347;4,619,942; 4,397,863; and 4,324,794 (applicants intend that thedisclosures of the compounds disclosed in these references areincorporated by reference herein in their entirety; all of thesecompounds may be provided with a single amidine group as opposed to twoamidine groups). Specific examples of such compounds are set forthbelow.

[0031] A first group of active agents useful for carrying out thepresent invention are bis-benzenes having the formula I:

[0032] wherein:

[0033] A and B are each independently selected from the group consistingof H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy,carboxy, and compounds of formula (i):

[0034] subject to the proviso that at least one of A and B is a compoundof formula (i);

[0035] R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;

[0036] R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl,cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen;

[0037] n is from 2 to 6; and

[0038] X is O, NH, or S;

[0039] or a pharmaceutically acceptable salt thereof. In one embodiment,R₁, R₂ and R₃ are H; X is O; and n is 5.

[0040] A second group of bis-benzenes that may be used to carry out thepresent invention are those having formula II:

[0041] wherein:

[0042] A and B are each independently selected from the group consistingof H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy,carboxy, and compounds of formula (i):

[0043] subject to the proviso that at least one of A and B is a compoundof formula (i);

[0044] R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;

[0045] R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl,cycloalkyl, aryl; aminoalkyl, alkylaminoalkyl or halogen;

[0046] X is linear or branched, saturated or unsaturated C1-C12 alkylcontaining up to 4 double bonds; or X is a heterocyclic aromatic groupselected from the group consisting of:

[0047] wherein

[0048] R₆, R₇, and R₈ are each independently selected from the groupconsisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, oroxyarylalkyl; R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl oralkylaminoalkyl;

[0049] or the pharmaceutically acceptable salts thereof.

[0050] A third group of compounds useful for carrying out the presentinvention are those having formula III:

[0051] wherein:

[0052] A and B are each independently selected from the group consistingof H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy,carboxy, and substituents of formula (i):

[0053] subject to the proviso that at least one of A and B is asubstituent of formula (i);

[0054] R₁ and R₂ are each independently selected from the groupconsisting of H., loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ groups on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;

[0055] R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl,cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen;

[0056] or two R₁ groups on the same amidine group together represent

[0057] wherein R₅ is

[0058] n is an integer from 0 to 2; and

[0059] A is a heterocyclic aromatic group selected from the groupconsisting of:

[0060] wherein

[0061] R₆, R₇, and R₈ are each independently selected from the groupconsisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, oroxyarylalkyl;

[0062] R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl oralkylaminoalkyl;

[0063] and the pharmaceutically acceptable salts thereof.

[0064] A fourth group of compounds useful for carrying out the presentinvention are those having formula IV:

[0065] wherein:

[0066] A and B are each independently selected from the group consistingof H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy,carboxy, and substituents of formula (i):

[0067] subject to the proviso that at least one of A and B is asubstituent of formula (i);

[0068] R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;

[0069] R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl,cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen;

[0070] or two R₁ groups on the same amidine group together represent

[0071] wherein R₅ is

[0072] and the pharmaceutically acceptable salts thereof

[0073] A fifth group of active compounds that may be used to carry outthe present invention are those having formula (V):

[0074] wherein:

[0075] A and B are each independently selected from the group consistingof H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy,carboxy, and substituents of formula (ii):

[0076] subject to the proviso that at least one of A and B is asubstituent of formula (ii);

[0077] R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl,halogen, oxyalkyl, oxyaryl, or oxyarylalkyl;

[0078] R₃ and R₄ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkylaryl, aryl, oxyaryl,aminoalkyl, aminoaryl, or halogen; and

[0079] each R₅ is independently selected from the group consisting of H,loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl,cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ toC₁₀ alkyl, hydroxyalkyl, or alkylene; and

[0080] R₆ is H, hydroxy, loweralkyl, alkoxyalkyl, hydroxyalkyl,aminoalkyl, alkylamino, alkylaminoalkyl, cycloalkyl, hydroxycycloalkyl,alkoxycycloalkyl, aryl, or alkylaryl;

[0081] or a pharmaceutically acceptable salt thereof.

[0082] A sixth group of active compounds of the present invention arethose having formula VI:

[0083] wherein:

[0084] A and B are each independently selected from the group consistingof H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo, hydroxy,carboxy, and substituents of formula (i):

[0085] subject to the proviso that at least one of A and B is asubstituent of formula (i);

[0086] R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R. group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;

[0087] R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl,cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen;

[0088] or two R₁ groups on the same amidine group together represent

[0089] wherein R₅ is

[0090] X is O, S or NH;

[0091] n is an integer from 1 to 8;

[0092] and the pharmaceutically acceptable salts thereof.

[0093] Salts of Active Compounds.

[0094] The active compounds may be converted to a corresponding acidaddition salt, or pharmaceutically acceptable salt, by virtue of thepresence of the nitrogen atoms.

[0095] The conversion is accomplished by treatment with at least astoichiometric amount of an appropriate acid, such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinicacid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicylic acid and the like. Typically,the free base is dissolved in an inert organic solvent such as diethylether, ethyl acetate, chloroform, ethanol or methanol and the like, andthe acid added in a similar solvent. The temperature is maintained at 0°-50° C. The resulting salt precipitates spontaneously or may be broughtout of solution with a less polar solvent.

[0096] The acid addition salts of the active compounds may be convertedto the corresponding free bases by treatment with at least astoichiometric amount of a suitable base such as sodium or potassiumhydroxide, potassium carbonate, sodium bicarbonate, ammonia, and thelike.

[0097] Utility and Testing.

[0098] The compounds are generally useful as described above. Thecompounds may be specifically tested in accordance with knowntechniques.

[0099] In one particular aspect, the invention relates to a method ofdetermining the presence of imidazoline receptors in vitro or in vivo inmammalian tissue, especially human tissue, by contacting the samplesuspected of containing such receptors with an active compound of theinvention and detecting the presence or absence of binding of suchcompound to a receptor in the sample. The detecting may be carried outby any suitable means, such as by labelling the active compound with adetectable group such as ³H, or by competitive displacement of anotherreceptor binding compound that it itself labelled, all in accordancewith known techniques. Detecting may be carried out with a library orcombinatorial library of compounds of the invention, as described below.

[0100] In another particular aspect,. the invention provides methods ofalleviating disease states as described above.

[0101] Potential for high selectivity and high affinity for imidazolinesites is determined in vitro by a modification of the method of Brown etal., Br. J.Pharmacology, Vol. 99, pp 481 varies as 486 (1990), asdescribed in Example II of U.S. Pat. No. 5,726,197 to Clark et al.

[0102] Lowering of intraocular pressure is shown in vivo by the methodof Moses, R. A., Tr. Am. Acad. Opth. and Otol., January-February 1962:88-95.

[0103] Potential for treatment of cerebral ischemia is shown in vitro bya modification of the method of Gotti et al., Brain Res., Vol 522(2), pp290-307 (1990).

[0104] The antihypertensive activity of the compounds may be determinedin conscious spontaneous hypertensive rats prepared with indwellingarterial catheter by the in vivo assay described in Popovic V. andPopovic P., J. Applied. Physiol., Vol. 15, pp. 727-728 (1960), or amodification thereof.

[0105] Administration.

[0106] In applying the compounds of this invention to treatment of theabove conditions, administration of the active compounds and saltsdescribed herein can be via any of the accepted modes of administration,including oral, parenteral and otherwise system of route ofadministration. Any pharmaceutically acceptable mode of administrationcan be used, including solid, semi-solid or liquid dosage forms, suchas, for example, tablets, suppositories, pills, capsules, powders,liquids, suspensions, or the like, preferably in unit dosage formssuitable for single administration of precise dosages, or in sustainedor controlled release dosage forms for the prolonged administration ofthe compound at a predetermined rate. The compositions will typicallyinclude a conventional pharmaceutical carrier or excipient and an activecompound or the pharmaceutically acceptable salts thereof and, inaddition, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, etc.

[0107] The amount of active compound administered will of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician. However, an effective dose for oral, parenteral and otherwisesystemic routes of administration is in the range of 0.01-20 mg/kg/day,preferably 0.1-10 mg/kg/day. For an average 70 kg human, this wouldamount to 0.7-1400 mg per day, or preferably 7-700 mg,/day.

[0108] For solid compositions, conventional non-toxic solid carriersinclude, for example, pharmaceutical grades of mannitol, lactose,cellulose, cellulose derivatives, sodium crosscarmellose, starch,magnesium stearate, sodium saccharin, talcum, glucose, sucrose,magnesium carbonate, and the like may be used. The active compound asdefined above may be formulated as suppositories using, for example,polyalkylene glycols, acetylated triglycerides and the like, as thecarrier. Liquid pharmaceutically administrable compositions can, forexample, be prepared by dissolving, dispersing, etc. an active compoundas defined above and optional pharmaceutical adjuvants in a carrier,such as, for example, water, saline, aqueous dextrose, glycerol,ethanol, and the like, to thereby form a solution or suspension. ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, for example,sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,sorbitan monolaurate, triethanolamine oleate, etc. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Reminigton's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. Thecomposition or formulation to be administered will, in any event,contain a quantity of the active compound(s) in an amount effective toalleviate the symptoms of the subject being treated.

[0109] Dosage forms or compositions containing active compounds or theirsalts) in the range of 0.25 to 95% with the balance made up fromnon-toxic carrier may be prepared.

[0110] For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, cellulose, cellulose derivatives, sodiumcrosscarmellose, starch, magnesium stearate, sodium saccharin, talcum,glucose, sucrose, magnesium, carbonate, and the like. Such compositionstake the form of solutions, suspensions, tablets, pills, capsules,powders, sustained release formulations and the like. Such compositionsmay contain 1%-95% active ingredient, more preferably 2-50%, mostpreferably 5-8%.

[0111] Parenteral administration is generally characterized byinjection, either subcutaneously, intramuscularly or intravenously.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate, triethanolamine sodium acetate, etc.

[0112] A more recently devised approach for parenteral administrationemploys tile implantation of a slow-release or sustained-release system,such that a constant level of dosage is maintained. See, e.g., U.S. Pat.No. 3,710,795.

[0113] The percentage of active compound contained in such parentalcomposition is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject. However,percentages of active ingredient of 0.1% to 10% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. Preferably thecomposition will comprise 0.2-2% of the active agent in solution.

[0114] In applying the compounds of the invention to treatment ofdiseases or disorders of the eye which are associated with an abnormallyhigh intraocular pressure, administration may be achieved by anypharmaceutically acceptable mode of administration which providesadequate local concentrations to provide the desired response. Theseinclude direct administration to the eye via drops and controlledrelease inserts or implants, as well as systemic administration aspreviously described.

[0115] Drops and solutions applied directly to the eye are typicallysterilized aqueous solutions containing 0.1% to 10%, most preferably0.5% to 1% of the active ingredient, along with suitable buffer,stabilizer, and preservative. The total concentration of solutes shouldbe such that, if possible, the resulting solution is isotonic with thelacrimal fluid (though this is not absolutely necessary) and has anequivalent pH in the, range of pH 6-8. Typical preservatives are phenylmercuric acetate, thimerosal, chlorobutanol, and benzalkonium chloride.Typical buffer systems and salts are based on, for example, citrate,borate or phosphate; suitable stabilizers include glycerin andpolysorbate 80. The aqueous solutions are formulated simply bydissolving the solutes in a suitable quantity of water, adjusting the pHto about 6.8-8.0, making a final volume adjustment with additionalwater, and sterilizing the preparation using methods known to those inthe art.

[0116] The dosage level of the resulting composition will, of course,depend on the concentration of the drops, the condition of the subjectand the individual magnitude of responses to treatment. However, atypical ocular composition could be administered at the rate of about2-10 drops per day per eye of a 0.5% solution of active ingredient.

[0117] The compositions of the present invention may also be formulatedfor administration in any convenient way by analogy with other topicalcompositions adapted for use in mammals. These compositions may bepresented for use in any conventional manner with the aid of any of awide variety of pharmaceutical carriers or vehicles. For such topicaladministration, a pharmaceutically acceptable non-toxic formulation cantake the form of semisolid, liquid, or solid, such as, for example,gels, creams, lotions, solutions, suspensions, ointments, powders, orthe like. As an example, the active components may be formulated into agel using ethanol, propylene glycol, propylene carbonate, polyethyleneglycols, diisopropyl adipate, glycerol, water, etc., with appropriategelling agents, such as Carbomers, Klucels, etc. If desired, theformulation may also contain minor amounts of non-toxic auxiliarysubstances such as preservatives, antioxidants, pH buffering agents,surface active agents, and the like. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in theart; for example, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., 16th Edition, 1980.

[0118] Combinatorial chemistry.

[0119] Numerous methods of synthesizing combinatorial libraries and(where desired) applying such molecules on solid supports (where themolecules may be either covalently or non-covalently bound, linked orattached to the solid support) are known, and such libraries can be madeand used in accordance with procedures known to those skilled in theart. See, e.g., J. Baldwin and I. Henderson, Recent Advances in theGeneration of Small-Molecule Combinatorial Libraries. Encoded SplitSynthesis and Solid-Phase Synthetic Methodology, Med. Res. Reviews 16,391-405 (1996), see also U.S. Pat. No. 5,565,324 to Still et al., U.S.Pat. No. 5,284,514 to Ellman et al., U.S. Pat. No. 5,445,934 to Fodor etal. (the disclosures of all United States patents cited herein are to beincorporated herein by reference in their entirety). Libraries of activecompounds as described herein (i.e., a group of compounds that have thegeneral definition of active compounds as described herein, but differfrom one another in specific structural features, patterns ofsubstitution(s), etc.), may be screened in accordance with knowntechniques to identify imidazoline receptor binding compounds withspecific desired characteristics.

[0120] The present invention is explained in greater detail in thefollowing non-limiting examples.

EXAMPLE 1

[0121] 1. Materials and mMthods

[0122] Membrane preparation. Membranes were prepared in accordance withknown techniques (R. Zonnenchein et al., Eur. J. Pharmacol. 190, 203-215(1990)). Briefly, male Sprague-Dawley rats (Hilltop, Scottsdale, Pa.,U.S.A.) weighing approximately 250-300 g were decapitated, their liversrapidly removed and immediately placed into 0.25 M cold sucrosesolution. The livers were weighed and minced with scissors before beinghomogenized at 4° C. in solution A (0.25 M sucrose containing 0.1 mMphenylmethyl sulfonyl fluoride 1:10v/v) for 1 min using a Biospechomogenizer (Bartlesville, Okla., U.S.A.). The homogenate wascentrifuged at 600×g for 10 min at 4° C. The resulting pellet was washedwith solution A, discarded and the supernatant fractions were combinedand centrifuged at 12,000×g for 30 min. This pellet was washed thenresuspended in cold buffer B (50 mM Tris/HCI pH 7.4). Using a BioRad(Hercules, Calif., U.S.A.) assay kit, the protein concentration wasdetermined using bovine serum albumin as the standard according to theassay of Bradford et al. (Bradford, M.M., 1976. Anal. Biochem. 72,248-254.). The preparation was stored at −80° C. at a concentration of20 mg protein /ml.

[0123] Saturation binding studies. Binding studies were performed intriplicate in 1 ml buffer B containing [³H]idazoxan (0.1-50 nM). Theaddition of rat liver membranes at a final concentration of 500 μgprotein /ml initiated binding, and the samples were incubated toequilibrium at 22° C. for 45 min. Non-specific binding was defined asthe amount of [³H]idazoxan bound in the presence of 10 μM cirazoline.

[0124] Competition binding studies. Competition experiments wereperformed in 1 ml buffer B containing 5 nM [3H]idazoxan. Increasingconcentrations of compound were added (1 nM -10 mM) and binding wasinitiated by the addition of rat liver membranes at a finalconcentration of 500 μg protein /ml. In all experiments, bound and freeradioligand were separated by vacuum filtration through Whatman GF/Cfilters and washed with 2×10 ml cold buffer B. Radioactivity on filterswas determined by liquid scintillation spectroscopy using a PackardTri-Carb 2100TR. Samples were run in triplicate and the results werederived from at least three experiments with the exception of Compounds4 and 8 which were run twice due to limited quantities.

[0125] Anti-Pneumocystis carinii activity. Induction, treatment andevaluation of P. carinii pneumonia in the rat was carried out accordingto methods described previously (Jones, S. K. et al., Antimicrob. AgentsChemother. 34, 1026-1030 (1990)). To summarize the scoring procedure,the mean histologic scores were determined by two examiners using ablinded protocol. P. carinii cysts were counted in the stained lungsections and assigned the following values; 0.5—less than 10 cystscounted per two fully examined sections; 1—scattered cysts with lessthan 10% of the lung tissue involved; 2—scattered cysts with limitedintense focal involvement and 10 to 25% of lung tissue involved;3—scattered cysts with numerous intense areas of focal involvement and26 to 50% of lung tissue involved; 4—cysts found throughout the tissuewith numerous intense focal areas of involvement having greater than 50%of lung tissue involved.

[0126] Chemical synthesis. The procedure for the synthesis of4-methoxybenzamidine was adapted from Tidwell et al. (J. Med. Chem. 33,1252-1257 (1990)). The procedure was altered in the following way; asolution of, 1,4dioxane was cooled to −10° C. and presaturated withhydrogen chloride (HCI) gas. 4Methoxybenzonitrile and anhydrous ethanolwere added and the reaction mixture was saturated with HCI gas. Thereaction flask was sealed and stirred at ambient temperature for twelvedays. The imidate was collected and dried under high vacuum for 20 minand then dissolved in ethanolic ammonia at −10° C. The reaction mixturewas stirred at room temperature overnight. The purification of4methoxybenzamidine was followed according to the published procedure.All other compounds were synthesized as previously described (B. Bergeret al., J. Pharmacol.Exp. Ther. 256, 883-889 (1991); Tidwell et al.,supra (1990)). Final compounds were analyzed using high-performanceliquid chromatography, proton nuclear magnetic resonance spectroscopyand elemental analysis.

[0127] Drugs. [³H]Idazoxan (specific activity 45 Ci/mmol) was purchasedfrom Amersham (Arlington Heights, Ill., U.S.A.), cirazoline fromResearch Biochemicals International (Natick, Mass., U.S.A.),phenylmethyl sulfonyl fluoride from Boelhringer Mannheim (Indianapolis,Ind., U.S.A.), sucrose from Fisher (Fair Lawn, N.J., U.S.A.) andTris/HCl from Schwartz/Mann Biotech (Cleveland, Ohio., U.S.A.).Benzamidine, yohimbine, phentolamine, epinephrine and bovine serumalbumin were purchased from Sigma (St. Louis, Mo., U.S.A.). 1,4-Dioxaneand 4-methoxybenzonitrile were purchased from Aldrich (Milwaukee, Wis.,U.S.A.), and HCl gas and ammonia from Matheson (Secaucus, N.J., U.S.A.).All other compounds were synthesized in our laboratory. Buffers wereprepared using deionized water from Dracor Water Systems (Kensington,Md., U.S.A.).

[0128] Data analysis. Binding analyses, statistics and graphics wereperformed using Prism 2.01 (Graphpad Software, Inc., San Diego, Calif.,U.S.A.). Binding parameters for inhibitors are given as mean ±S.D.except Compounds 4 and 8 which are expressed as mean ±95% confidenceintervals due to limited supply of compound. The calculated log of theoctanol:water partition coefficient (ClogP) was calculated using MedChemSoftware 3.5 (Daylight Chemical Information Systems, New Orleans, La.,U.S.A.).

[0129] 2. Results

[0130] [³H]Idazoxan saturation binding in rat liver membranes. Rat livermembranes, prepared according to the method of Zonienschein et al.(supra) were reported to be rich in imidazoline I₂ sites while excluding(α₂-adrenoceptors. Saturation binding analyses reported a B_(max) of438±30 fmol/mg protein with non-specific binding representing 10-15% oftotal binding, which conforms with the results reported by Zonnenscheinet al. (1990). [³ H]Idazoxan bound the rat liver membranes with aK_(d)=14±2 nM for saturable binding. To confirm that α₂-adrenoceptorswere absent from the preparation, displacement experiments wereperformed using yohimbine, a selective α₂-adrenoceptor antagonist;epinephrine, a selective α₂-adrenoceptor agonist; phentolamine, anα₂-adrenoceptor antagonist; and cirazoline, an α₂-adrenoceptorantagonist as competitive inhibitors of [³H]idazoxan binding. FIG. 1shows that selective α₂-adrenoceptor ligands did not compete forradioligand binding, whereas phentolamine displaced [³H] idazoxanbinding with low affinity (K_(i)>5000), and cirazoline with highaffinity (K_(i)=3.0±0.4 nM).

[0131] Competition binding studies. A competitive inhibition curve usingpentamidine as the inhibitor of [³H]idazoxan binding was generated (datanot shown). The K_(i) of 1.4±0.22 nM demonstrates that pentamidine bindsto imidazoline I₂ sites with very high affinity, comparable to that ofthe most potent ligands. Several interesting structure/activityrelationships were observed from the data in Table 1. Taking pentamidine(Compound 1) as the. lead compound, no change in affinity was observedwhen the central alkyl link was increased to six carbons (Compound 2) orthe amidine groups replaced with imidazoline moieties (Compound 3).Moderate reductions in affinity (10-100 nM) were observed when the alkylchain was shortened further (Compounds 4 and 7) and a similar reductionwas noted when one of the cations was replaced by a nitro group(Compound 8). Loss of one benzamidine moiety (Compound 9) caused afurther reduction in affinity; however, when both the amidine phenoxymoiety and four of the carbons from the alkyl chain were removed frompentamidine (Compound 10), there was no further reduction in affinitywhen compared to Compound 9.

[0132] Interestingly, methoxy substitution meta to the cation (Compounds5 and 6) produced varied results depending on the nature of the cationicmoiety. When compared to the unsubstituted analog (Compound 7), methoxysubstitution (Compound 6) resulted in over a two log decrease inimidazoline receptor binding. However, methoxy substitution of theimidazoline analog (Compound 5) resulted in a compound with similaraffinity to Compound 7. The low affinity of Compound 6 may be a resultof its very low ClogP. Likewise, a comparison of Compounds 10-12 showsthat Compound 11 with the lowest ClogP of all the compounds evaluatedalso had the lowest affinity. The low affinity of Compound 11 may be dueto the existence of contributing resonance structures to form azwitterion The basicity and therefore the binding property of theamidines are greatly influenced by the presence of differentsubstituents on the benzamidine ring. Both benzamidine (Compound 12) and4-methoxybenzamidine (Compound 10) display resonance stabilization ofthe positive charge throughout the molecule; however, the phenoxide ionof 4-hydroxybenzamidine neutralizes the positive charge, thereforemaking it unavailable for receptor binding. This molecule lends supportto the theory that a strong cation is an important factor in receptorbinding.

[0133] ClogP. ClogP increases as compound lipophilicity increases. Table1 shows that Compound 3 is the most lipophilic (ClogP=6.0) and Compound11 is the least lipophilic (ClogP=0.08) of the compounds tested. Thereis no significant correlation for the group as a whole between K_(i) andClogP; Pearson correlation coefficient r=−0.38 P=0.22. However, it isinteresting to note that the ClogP and K_(i) values for Compounds 5 and6 differ considerably. The low affinity of Compound 6 (K_(i)≅3500 nM)may be attributed to its hydrophilic nature, denoted by the very lowClogP (0.09). Similarly, when Compounds 10-12 are compared, thederivative with the lowest ClogP (Compound 11) shows weakest affinityfor the imidazoline I₂ site (K_(i)≅30,000 nM, ClogP=0.08). TABLE 1Affinity for I₂ binding sites, anti-P. carinii activity and ClogP valuesfor pentamidine (Compound 1) and various analogs. Structure K_(i)(nM)Hill Slope ClogP 1

 1.4 ± 0.22 −0.8 2.3 2

 3.2 ± 0.36 −1.2 2.8 3

 4.3 ± 1.09 −0.8 6.0 4

23.4 ± 6.8  −0.8 1.8 5

50.1 ± 1.06 −0.9 4.0 6

˜3500 −0.9 .09 7

27.2 ± 7.63 −0.8 1.3 8

62.0 ± 25   −0.8 3.7 9

 319 ± 39.5 −0.8 .8 10

 228 ± 36.5 −0.8 0.7 11

˜30,000 −0.3 .08 12

 580 ± 61.3 −0.8 0.6 # are stated as mean Ki ± 95% confidence intervals.

[0134] Anti-Pneumocystis carinii activity. Pentamidine exhibits anti-P.carinii activity with a mean histologic score of 1.3. This is amoderately effective antimicrobial agent, having activity against avariety of organisms (B. Blagburn et al., Antimicrob. Agents Chemother.35, 1520-1523 (1991); C. Bell et al., Antimicrob. Agents Chemother. 35,1099-1107 (1991); D. Lindsay et al., Antimicrob. Agents Chemother. 35,1914-1916 (1991)). Five of the compounds have anti-P. carinii activitygreater than pentamidine (Compounds 2,3,4,5,6), while 2 of the compoundshave no activity at all (Compounds 9,11). Compounds 5 and 6 are equallyeffective anti-P. carinii agents, yet their respective affinities forthe imidazoline I₂ site differ 70-fold. These compounds provide anexample that affinity for the imidazoline I₂ site does not correlate toanti-P. carinii activity; Pearson correlation coefficient r=0.60 P=0.09.

EXAMPLE 2 AND 3

[0135] The compounds set forth in Table 2 and Table 3 below, which areeither known and can be synthesized in accordance with known techniquesas described above or variations thereof which will be apparent to thoseskilled in the art, are also illustrative of the present invention.TABLE 2 IMIDAZOLNE RECEPTOR BINDING COMPOUNDS Compound Structure Ki (nM)001 KAO 011

158.0 BABIM

7,512 BABB

5,526 BIBB

44,780 DB 205

346,700 DB 183

65,640 003 KAO 111

44,700 DAP 092

18,090 DB 75

653 DB 60

630 DB 181

5,503 DB 244

6,755 DB 103

236.0 DB 351

110.6 DB 262

3,785

[0136] TABLE 3 IMIDAZOLINE RECEPTOR BINDING COMPOUNDS Compound StructureKi (nM) MC 96

1.4 FS 44

2.8 FS 104

1.5 FS 113

2.2 MC97c

1215 FS 117

32.7

[0137] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

We claim:
 1. A method of binding the imidazoline receptor, comprising:contacting a bis-benzene to said imidazoline receptor in an amounteffective to bind to said receptor, wherein said bis-benzene contains atleast one amidine group.
 2. A method according to claim 1, wherein saidcontacting step is carried out in vitro.
 3. A method according to claim1, wherein said contacting step is carried out in vitro with cells thatexpress said imidazoline receptor.
 4. A method according to claim 1,wherein said contacting step is carried out in vitro with a cell-freepreparation comprising said imidazoline receptor.
 5. A method accordingto claim 1, wherein said contacting step is carried out in vivo.
 6. Amethod according to claim 1, wherein said contacting step is carried outin vivo by administering said compound to a subject afflicted with adisease state which is alleviable by treatment with a compound havinghigh selectivity and affinity for the imidazoline receptor site.
 7. Amethod according to claim 1, wherein said bis-benzene has the formula I:

wherein: A and B are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo,hydroxy, carboxy, and compounds of formula (i):

subject to the proviso that at least one of A and B is a compound offormula (i); R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen; n is from 2 to 6; and X isO, NH, or S; or a pharmaceutically acceptable salt thereof.
 8. A methodaccording to claim 7, wherein R₁, R₂ and R₃ are H; wherein X is O; andwherein n is
 5. 9. A method according to claim 1, wherein saidbis-benzene has the formula II:

wherein: A and B are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo,hydroxy, carboxy, and compounds of formula (i):

subject to the proviso that at least one of A and B is a compound offormula (i); R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two RI group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen; X is linear or branched,saturated or unsaturated C1-C12 alkyl containing up to 4 double bonds;or X is a heterocyclic aromatic group selected from the group consistingof:

wherein R₆, R₇, and R₈ are each independently selected from the groupconsisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, oroxyarylalkyl; R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl oralkylaminoalkyl; or the pharmaceutically acceptable salts thereof.
 10. Amethod according to claim 1, wherein said bis-benzene has the formulaIII:

wherein: A and B are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo,hydroxy, carboxy, and substituents of formula (i):

subject to the proviso that at least one of A and B is a substituent offormula (i); R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ groups on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen; or two R₁ groups on thesame amidine group together represent

wherein R₅ is

n is an integer from 0 to 2; and A is a heterocyclic aromatic groupselected from the group consisting of:

wherein R₆, R₇, and R₈ are each independently selected from the groupconsisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, oroxyarylalkyl; R₉ is hydrogen, loweralkyl, hydroxy, aminoalkyl oralkylaminoalkyl; and the pharmaceutically acceptable salts thereof. 11.A method according to claim 1, wherein said bis-benzamidine has theformula IV:

wherein: A and B are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo,hydroxy, carboxy, and substituents of formula (i):

subject to the proviso that at least one of A and B is a substituent offormula (i); R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on thesame amidine group together represent —(CH₂)m—wherein m is 2, 3, or 4;R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen; or two R₁ groups on thesame amidine group together represent

wherein R₅ is

and the pharmaceutically acceptable salts thereof.
 12. A methodaccording to claim 1, said bis-benzene having the formula (V): B

wherein: A and B are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo,hydroxy, carboxy, and substituents of formula (ii):

subject to the proviso that at least one of A and B is a substituent offormula (ii); R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, aryl, alkylaryl, aminoalkyl, aminoaryl,halogen, oxyalkyl, oxyaryl, or oxyarylalkyl; R₃ and R₄ are eachindependently selected from the group consisting of H, loweralkyl,oxyalkyl, alkylaryl, aryl, oxyaryl, aminoalkyl, aminoaryl, or halogen;and each R₅ is independently selected from the group consisting of H,loweralkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl,cycloalkyl, aryl, or alkylaryl or two R₅ groups together represent C₂ toC₁₀ alkyl, hydroxyalkyl, or alkylene; and R₆ is H, hydroxy, loweralkyl,alkoxyalkyl, hydroxyalkyl, aminoalkyl, alkylamino, alkylaminoalkyl,cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aryl, or alkylaryl; ora pharmaceutically acceptable salt thereof.
 13. A method according toclaim 1, wherein said bis-benzene has the formula VI:

wherein: A and B are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, nitro, amino, aminoalkyl, halo,hydroxy, carboxy, and substituents of formula (i):

subject to the proviso that at least one of A and B is a substituent offormula (i); R₁ and R₂ are each independently selected from the groupconsisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cyloalkyl, aryl,hydroxyalkyl, aminoalkyl and alkylaminoalkyl; or two R₁ group on thesame amidine group together represent —(CH₂)m- wherein m is 2, 3, or 4;R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen; or two R₁ groups on thesame amidine group together represent

wherein R₅ is

X is O, S or NH; n is an integer from 1 to 8; and the pharmaceuticallyacceptable salts thereof
 14. A method of identifying imidazolinereceptor binding agents, comprising the steps of: providing a library ofbis-benzene compounds, said bis-benzene compound containing at least oneamidine group; and screening said library for compounds that bind tosaid imidazoline receptor.
 15. A method according to claim 14, whereinsaid library is a combinatorial library.
 16. A method according to claim14, wherein said bis-benzene compounds are immobilized on a solidsupport.